Growth factors are polypeptides which stimulate a wide variety of biological responses (e.g., DNA synthesis, cell division, expression of specific genes, etc.) in a defined population of target cells. A variety of growth factors have been identified, including transforming growth factor .beta.1 (TGF-.beta.1), TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, TGF-,.beta.5, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-1) and IGF-II.
IGF-I and IGF-II are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7.5 kilodaltons (kD). IGF-I mediates the major effects of growth hormone, and thus is the primary mediator of growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in fetal growth. Both IGF-I and IGF-II have insulin-like activities (hence their names), and are mitogenic (stimulate cell division) for the cells in neural tissue, muscle, reproductive tissue, skeletal tissue and a wide variety of other tissues.
Unlike most growth factors, the IGFs are present in substantial quantity in the circulation, but only a very small fraction of this IGF is free in the circulation or in other body fluids. Most circulating IGF is bound to an IGF-binding protein called IGFBP-3. IGF-I may be measured in blood serum to diagnose abnormal growth-related conditions, e.g., pituitary gigantism, acromegaly, dwarfism, various growth hormone deficiencies, etc. Although IGF-I is produced in many tissues, most circulating IGF-I is believed to be synthesized in the liver.
Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF-I or IGF-II, IGFBP-3, and a larger protein subunit termed the acid labile subunit (ALS). This ternary complex is composed of equimolar amounts of each of the three components. ALS has no direct IGF binding activity and appears to bind only to the IGF/IGFBP-3 binary complex. The ternary complex of IGF+IGFBP-3 +ALS has a molecular weight of approximately 150 Kd. This ternary complex is alleged to function in the circulation "as a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes in the concentration of free IGF" (Blum et al., 1991, "Plasma IGFBP-3 Levels as Clinical Indicators" in Modem Concepts in Insulin-Like Growth Factors, E. M. Spencer, ed., Elsevier, New York, pp. 381-393). The ternary complex is also believed to play an important role in the prevention of hypoglycemia due to high doses of IGFI, by binding IGF-I/IGFBP-3 complex and restricting its distribution (Zapf et al., 1994, "Intravenously Injected Insulin-like Growth Factor (IGF) I/IGF Binding Protein-3 Complex Exerts Insulin-like Effects in Hypophysectomized, but Not in Normal Rats", Clinical Investigation 95: 179-186). ALS is growth hormone-dependent, so hypophysectomized rats and other subjects with insufficient levels of growth hormone have little to noALS (Baxter, 1990, 1990, "Circulating Levels and Molecular Distribution of the Acid-labile (.alpha.) Subunit of the High Molecular Weight Insulin-like Growth Factor-Binding Protein Complex" J Clin. Endocrinol. 70(5): 1347-1353).
Nearly all of the IGF-I, IGF-II and IGFBP-3 in the circulation is in complexes, so very little free IGF is detectable. Moreover, a high level of free IGF in blood is undesirable. High blood levels of free IGF lead to serious hypoglycemia, due to the insulin-like activities of IGF, as well as other adverse side effects. In contrast to the IGFs and IGFBP-3, there is a substantial pool of free ALS in plasma which assures that IGF/IGFB-3 complex entering the circulation immediately forms the ternary complex. However, it is believed that saturating free ALS by administration of high levels of IGF-I/IGFBP-3 will also lead to hypoglycemia (Zapf et al., ibid).
IGFBP-3 is the most abundant IGF binding protein in the circulation, but at least five other distinct IGF binding proteins (IGFBPs) have been identified in various tissues and body fluids. Although these proteins bind IGFs, they each originate from separate genes and have distinct amino acid sequences. Thus, the binding proteins are not merely analogs or derivatives of a common precursor. Unlike IGFBP-3, the other IGFBPs in the circulation are not saturated with IGFs. None of the IGFBPs other than IGFBP-3 can form the 150 Kd ternary complex with IGF-I and ALS.
IGF-I and IGFBP-3 may be purified from natural sources or produced by recombinant means. For instance, purification of IGF-I from human serum is well known to the art (Rinderknecht et al., 1976, Proc. Natl. Acad. Sci, (USA) 73: 2365-2369). Production of IGF-I by recombinant processes is shown in EP 0,128,733, published in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et al., (1986, "Growth Hormone-Dependent Insulin-Like Growth Factors (IGF) Binding Protein from Human Plasma Differs from Other Human IGF Binding Proteins", Biochem Biophys. Res, Comm, 139: 1256-1261). IGFBP-3 may be synthesized by recombinant organisms as discussed in Sommer et al. (1991, "Molecular Genetics and Action of Recombinant Insulin-Like Growth Factor Binding Protein-3", in Modem Concepts of Insulin-Like Growth Factors, E. M. Spencer, ed., Elsevier, New York, pp. 715-728). This recombinant IGFBP-3 binds IGF-I in a 1:1 molar ratio.
Studies with IGF-I have suggested its utility in treating a wide variety of indications. Clemmons and Underwood (1994, "Uses of Human Insulin-like Growth Factor-I in Clinical Conditions" J Clin. Endocrinol. Metabol. 79(1): 4-6) have suggested that IGF-I may be useful for the treatment of catabolic states, such as can arise due to trauma, severe burns, and major surgery. Clemmons and Underwood (supra) also suggest the utility of IGF-I in the treatment of acute and chronic renal disorders. IGF-I may be useful for the treatment of lymphopoietic disorders (Clark et al., 1993, "Insulin-like Growth Factor I Stimulation of Lymphopoiesis" J Clin. Invest. 92: 540-548). IGF-I has also been suggested as potentially useful in the treatment of bone disorders, such as osteoporosis, as well as wound healing and peripheral nerve disorders (Delany et al., 1994, "Cellular and Clinical Perspectives on Skeletal Insulin-like Growth Factor I" J. Cell. Biochem. 55(3): 328-333; Steenfos, 1994, "Growth Factors and Wound Healing" Scand J Plast. Reconstr. Surg. Hand Surg. 28(2): 95-105; Lewis et al., 1993, "Insulin-like Growth Factor I: Potential for Treatment of Motor Neuronal Disorders" Exp. Neurol. 124(1): 73-88).
IGF-I, when administered alone, can give rise to multiple deleterious side effects. The most commonly cited side effect of IGF-I administration is the induction of hypoglycemia. IGF-I induces significant hypoglycemia (significant hypoglycemia is normally defined as a decrease in blood glucose of 30% or more) in humans at doses of 30 .mu.g/kg by intravenous administration and 100 .mu.g/kg by subcutaneous administration (Lieberman et al., 1992, "Effects of Recombinant Human Insulin-like Growth Factor-I (rhIGF-I) on Total and Free IGF-I Concentrations, IGF-Binding Proteins, and Glycemic Response in Humans", J. Clin. Endocrinol. Metab. 75(1): 30-36; Guler et al., 1987, "Short-term Metabolic Effects of Recombinant Human Insulin-like Growth Factor I in Healthy Adults", New England J. Med. 317(3): 137-140). Other side effects include hypophosphatemia, which can cause muscle seizures and cardiac arrhythmia, and changes in sodium excretion, which can lead to edema.
The activities of the IGF-I/IGFBP-3 complex have been less extensively studied. In wound healing, topical administration of IGF-I/IGFBP-3 complex to rat and pig wounds is significantly more effective for promoting wound healing than administration of IGF-I alone (Sommer et al., supra).
Some studies have been performed using systemically administered IGF-I/IGFBP-3 complex, although usually at low doses. Zapf et al. (supra) gave normal and hypophysectomized rats a 4 mg/kg IV bolus of IGF-I/IGFBP-3 complex. This dose induced significant hypoglycemia in hypophysectomized rats, which are deficient in growth hormone and growth hormone-dependent proteins (such as ALS), but not normal rats. Sommer et al. (supra) gave a greater dose of IGF-I/IGFBP-3 complex to hypophysectomized rats, 40 mg/kg. This dose, which was administered by subcutaneous bolus injection, induced significant hypoglycemia (50% reduction in blood glucose). Although the dose given by Sommer et al. appears to be significantly greater than that given by Zapf et al., Sommer used a different route of administration (subcutaneous). Subcutaneous administration normally results in a lower and delayed peak concentration in the blood, particularly with large protein drugs (for example, see Goth's Medical Pharmacology, 13th edition, Clark, W. G., Brater, D. C., and Johnson, A. R., eds. Mosby Year Book, St. Louis, 1992 and Goodman and Gilman's The Pharmacological Basis of Therapeutics, Eighth edition, Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P., eds., Pergamon Press, New York, 1990).
In addition to testing IGF-I and IGF-I/IGFBP-3 complex, Zapf also forwarded a model for explaining why IGF-I/IGFBP-3 complex causes a lower degree of hypoglycemia compared to IGF-I alone. The Zapf model is the first and only model that can be used to make quantitative predictions as to the dose of IGF-I/IGFBP-3 complex that will cause hypoglycemia. The Zapf model predicts that IGF-I/IGFBP-3 complex bound in the ternary complex with ALS is non-hypoglycemic, but IGF-LIGFBP-3 complex in the 40 kD complex (i.e., not bound to ALS) can cause hypoglycemia. IGF-I alone is hypoglycemic because there is no excess IGFBP-3 to form the 40 kD complex, and thus free IGF-I cannot form the 150 kD ternary complex (Baxter and Martin, 1989, "Structure of the M.sub.r 140,000 Growth Hormone-dependent Insulin-like Growth Factor Binding Protein Complex: Determination by Reconstitution and Affinity-labeling" Proc. Natl. Acad Sci. USA 86: 6898-6902)., The Zapf model predicts that adding enough IGF-I/IGFBP-3 complex to saturate ALS in the blood would lead to hypoglycemia. This model is supported by Baxter et al., who suggest that low ALS levels are cause of hypoglycemia in patients with nonislet cell tumors (1995, "Regulation of the Insulin-like Growth Factors and Their Binding Proteins by Glucocorticoid and Growth Hormone in Nonislet Cell Tumor Hypoglycemia" J. Clin. Endocrinol. Metabol. 80(9): 2700-2708).
The model disclosed in Zapf may be used to calculate the dose at which IGF-I/IGFBP-3 complex is expected to induce hypoglycemia. This calculation requires the determination of (a) the amount of IGF-I/IGFBP-3 complex required to bind all the free ALS in the blood and (b) the amount of IGF-I/IGFBP-3 complex that is the molar equivalent of the dose of free IGF-I that induces hypoglycemia. These two numbers are added together to find the dose of IGF-I/IGFBP-3 complex that is expected to induce hypoglycemia.
ALS levels, both total and free ALS, have been measured in humans and rats (Baxter, supra; Baxter and Dai, 1994, "Purification and Characterization of the Acid-labile Subunit of Rat Serum Insulin-like Growth Factor Binding Complex" Endocrinol. 134(2): 848-852). In rats, total ALS is reported to be 42 .mu.g/ml in blood (ALS is limited to the vascular space due to its large size). Of that 42 .mu.g/ml, 33.6 .mu.g/ml (80%) of the ALS is free (i.e., not bound to the IGFI/IGFBP-3 complex) (Baxter and Dai, supra). In normal humans, total ALS is 24.2 .mu.g/ml, with one third, or 8 .mu.g/ml, of the total as free ALS. The amount of IGF-I/IGFBP-3 complex required to bind the free ALS is the molar equivalent of the free ALS; approximately 16.5 .mu.g/ml in the rat and 4 .mu.g/ml in the human. These numbers are then multiplied by the blood volumes of rats and humans, respectively, to yield the quantity of IGF-I/IGFBP-3 complex required to bind all of the free ALS (total blood volume is 54 ml/kg in rats, 74.3 ml/kg in humans (Davies and Morris, 1993, "Physiological Parameters in Laboratory Animals and Humans", Pharm. Res. 10(7): 1093-1095). Thus, the amount of IGF-I/IGFBP-3 complex required to bind all of the free ALS is 891 .mu.g/kg in rats and 300 .mu.g/kg in humans. Significant hypoglycemia is induced by IV IGFI at 0.8 mg/kg in rats (Zapf et al., supra) and 0.03 mg/ml in humans Lieberman et al., supra). The molar equivalent amounts of IGF-I/IGFBP3 complex are 4 mg/kg and 0.15 mg/kg, respectively. Thus, the amount of IGF-I/IGFBP-3 complex expected to produce hypoglycemia, administered IV, is 4.9 mg/kg in rats and 0.45 mg/kg in humans.
It would be desirable to give doses of IGF-I/IGFBP-3 complex that are even greater than the doses that are predicted to cause hypoglycemia. This is because of the expected greater efficacy of a higher dose. Studies with IGF-I/IGFBP-3 complex show a dose-response relationship, but show no signs of a plateau in the response to increasing amounts of the complex, suggesting that greater doses would lead to increased efficacy (Bagi et al., 1994, "Benefit of Systemically Administered rhlGF-I and rhlGF-I/IGFBP-3 on Cancellous Bone in Ovariectomized Rats", J Bone Mineral Res. 9(8): 1301-1311; Bagi et al., 1995, "Systemic Administration of rhlGF-I or rhIGF-I/IGFBP-3 Increases Cortical Bone and Lean Body Mass in Ovariectomized Rats", Bone 16(4 suppl.): 263S-269S; Bagi et al., 1995, "Treatment of Ovariectomized Rats with the Complex of rhIGF-I/IGFBP-3 Increases Cortical and Cancellous Bone Mass and Improves Structure in the Femoral Neck". Calcif Tiss. Int. 57: 40-46).
Accordingly, there exists in the art a need for a method for providing high dose IGF-I or IGF-I/IGFBP-3 complex therapy.